1. Field of the Invention
The invention relates to the prevention and/or treatment of neural tissue damage resulting from ischemia and reperfusion injury. More particularly, the invention concerns the prevention or treatment of vascular stroke, other neurodegenerative diseases and occlusion of coronary arteries, by administering selective inhibitors of the nucleic enzyme poly(adenosine 5xe2x80x2-diphospho-ribose) polymerase [xe2x80x9cpoly(ADP-ribose) polymerasexe2x80x9d or xe2x80x9cPARPxe2x80x9d, which is also sometimes called xe2x80x9cPARSxe2x80x9d for poly(ADP-ribose) synthetase].
2. Description of the Prior Art
Poly(ADP-ribose) polymerase (xe2x80x9cPARPxe2x80x9d) is an enzyme located in the nuclei of cells of various organs, including muscle, heart and brain cells. PARP plays a physiological role in the repair of strand breaks in DNA. Once activated by damaged DNA fragments, PARP catalyzes the attachment of up to 100 ADP-ribose units to a variety of nuclear proteins, including histones and PARP itself. While the exact range of functions of PARP has not been established, this enzyme is thought to play a role in enhancing DNA repair.
During major cellular stresses, however, the extensive activation of PARP can rapidly lead to cell death through depletion of energy stores. Four molecules of ATP are consumed for every molecule of NAD (the source of ADP-ribose) regenerated. Thus, NAD, the substrate of PARP, is depleted by massive PARP activation and, in the efforts to re-synthesize NAD, ATP may also be depleted.
Neural damage following stroke and other neurodegenerative processes is thought to result from a massive release of the excitatory neurotransmitter glutamate, which acts upon the N-methyl-D-aspartate (NMDA) receptors and other subtype receptors. Evidence includes findings in many animal species, as well as in cerebral cortical cultures treated with glutamate or NMDA, that glutamate receptor antagonists block neural damage following vascular stroke. Dawson et al., xe2x80x9cProtection of the Brain from Ischemiaxe2x80x9d, Cerebrovascular Disease, 319-325 (ed. Batjer 1997).
The stimulation of NMDA receptors, in turn, activates the enzyme neuronal nitric oxide synthase (nNOS), which causes the formation of nitric oxide (NO), which more directly mediates neurotoxicity. Protection against NMDA neurotoxicity has occurred following treatment with NOS inhibitors. See Dawson et al., xe2x80x9cNitric Oxide Mediates Glutamate neurotoxicity in Primary Cortical Culturesxe2x80x9d, Proc. Natl. Acad. Sci. USA, 88:6368 (1991); and Dawson et al., xe2x80x9cMechanisms of Nitric Oxide-mediated Neurotoxicity in Primary Brain Culturesxe2x80x9d, J. Neurosci., 13:2651-61 (1993). Protection against NMDA neurotoxicity can also occur in cortical cultures from mice with targeted disruption of nNOS. See Dawson et al., xe2x80x9cResistance to Neurotoxicity in Cortical Cultures from Neuronal Nitric Oxide Synthase Deficient Micexe2x80x9d, J. Neurosci., 16:2479-87 (1996). It is known that neural damage following vascular stroke is markedly diminished in animals treated with NOS inhibitors or in mice with nNOS gene disruption. Iadedcola, xe2x80x9cBright and Dark Sides or Nitric Oxide in Ischemic Brain Injuryxe2x80x9d, Trends Neurosci., 20:132-39 (1997); and Huang et al., xe2x80x9cEffects of Cerebral Ischemia in Mice Deficient in Neuronal Nitric Oxide Synthasexe2x80x9d, Science, 265:1883-85 (1994).
FIG. 5 provides a simple model of the following sequence of the multitude of cellular events that presumably takes place in the PARP activation associated with ischemia:
(1) Ischemia following blood vessel occlusion reduces the resting membrane potential of glia and neurons in the tissue.
(2) Potassium leaks out of cells and depolarizes the neurons, leading to a massive release of glutamate.
(3) Acting via NMDA receptors, glutamate triggers a release of NO, which combines with superoxide to form peroxynitrite.
(4) Peroxynitrite damages DNA, fragments of which then activate PARP.
(5) Massive activation of PARP depletes NAD via ADP-ribose polymer formation.
(6) ATP is depleted in an effort to re-synthesize NAD, leading to cell death by energy depletion.
NO is a free radical that reacts chemically with multiple cellular targets to elicit a range of activities from cellular signalling to cell death. Most of the toxic effects of NO appear to be a result of the reaction of NO with superoxide to form the extremely toxic peroxynitrite. See Beckman et al., xe2x80x9cPathological Implications of Nitric Oxide, Superoxide and Peroxynitrite Formationxe2x80x9d, Biochem. Soc. Trans., 21:330-34 (1993). Either NO or peroxynitrite can cause DNA damage, which activates PARP. PARP activation plays a key role in both NMDA- and NO-induced neurotoxicity, as shown by the use of PARP inhibitors to prevent such toxicity in cortical cultures in proportion to their potencies as inhibitors of this enzyme (Zhang et al., xe2x80x9cNitric Oxide Activation of Poly(ADP-ribose) Synthetase in Neurotoxicityxe2x80x9d, Science, 263:786-89 (1994)) and in hippocampal slices (Wallis et al., xe2x80x9cNeuroprotection Against Nitric Oxide Injury with Inhibitors of ADP-ribosylation, Neuroreport, 5:313, 245-48 (1993)). Zhang et al., U.S. Pat. No. 5,587,384 issued Dec. 24, 1996 also discusses the use of PARP inhibitors, such as benzamide and 1,5-dihydroxyisoquinoline, to prevent NMDA-mediated neurotoxicity and, thus, treat stroke, Alzheimer""s disease, Parkinson""s disease and Huntingtin""s disease.
Using this model, the conventional thought that neurotoxicity came from glutamate acting through NO has suggested that the modest protective effects of non-selective PARP inhibitors, which are comparable to the protective effects of inhibitors of NO formation or drugs that block glutamate receptors, were the best one could reasonably expect.
The NMDA-NO model, however, provided only one potential mechanism for neural injury such as stroke. There has been substantial evidence that other mechanisms, such as the production of oxygen-free radicals, independently of nitric oxide, also play a role. For example, PARP activation has been shown to provide an index of damage following neurotoxic insults, not only by glutamate (via NMDA receptor stimulation) and reactive oxygen intermediates, but also by amyloid xcex2-protein, n-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its active metabolite N-methyl-4-phenylpyridine (MPP*), which participate in such pathological conditions as stroke, Alzheimer""s disease and Parkinson""s disease. Zhang et al., xe2x80x9cPoly(ADP-Ribose) Synthetase Activation: An Early Indicator of Neurotoxic DNA Damagexe2x80x9d, J. of Neurochem., 65:3, 1411-14 (1995). See also, Choi, xe2x80x9cGlutamate Neurotoxicity and Diseases of the Nervous system, Neuron, 1:623-34 (1988); and Meldrum et al., xe2x80x9cExcitatory Amino Acid Neurotoxicity and Neurodegenerative Diseasexe2x80x9d, Trends in Pharmacological Sciences, 11:379-87 (1990); Choi et al., xe2x80x9cThe Role of Glutamate Neurotoxicity in Hypoxic ischemic Neuronal Deathxe2x80x9d, Ann. Rev. of Neurosci., 13:171-78 (1990). Thus, the relative contribution of oxygen-free radicals versus the NO system has been somewhat unclear.
It has been demonstrated that single injections of PARP inhibitors have reduced the infarct size caused by ischemia and reperfusion of the heart or skeletal muscle in rabbits. In these studies, a single injection of the non-selective PARP 3-aminobenzamide (10 mg/kg), either one minute before occlusion or one minute before reperfusion, caused similar reductions in infarct size in the heart (32-42%). Another PARP inhibitor, 1,5-dihydroxyisoquinoline (1 mg/kg), reduced infarct size by a comparable degree (38-48%). Thiemermann et al., xe2x80x9cInhibition of the Activity of Poly(ADP Ribose) Synthetase Reduces Ischemia-Reperfusion Injury in the Heart and Skeletal Musclexe2x80x9d, Proc. Natl. Acad. Sci. USA, 94:679-83 (1997). This finding has suggested that PARP inhibitors might be able to salvage previously ischemic heart or skeletal muscle tissue.
However, the approach of using PARP inhibitors generally to reduce NMDA-receptor stimulation or to treat or prevent tissue damage caused by NO is limited in effect. Accordingly, there remains a need for a procedure that produces a more potent and reliable effect downstream of the NMDA-NO sequence of bioevents by using an inhibitor that is selective of PARP activity itself, as opposed to using a non-selective PARP inhibitor that could exert many other non-specific actions, including depression of upstream events, such as NMDA-receptor activation and/or NO production.
The occurrence of side effects observed with non-selective PARP inhibitors are discussed in Milam et al., xe2x80x9cInhibitors of Poly(Adenosine Diphosphate-Ribose) Synthesis: Effect on Other Metabolic Processes,xe2x80x9d Science, 223:589-91 (1984). Specifically, the non-selective PARP inhibitors 3-aminobenzamide and benzamide not only inhibited the action of PARP but also were shown to affect cell viability, glucose metabolism, and DNA synthesis. Thus, it was concluded, the usefulness of these particular PARP inhibitors may be severely restricted by the difficulty of finding a dose small enough to inhibit the enzyme without producing additional metabolic effects. Banasik et al., in xe2x80x9cSpecific Inhibitors of Poly(ADP-Ribose) Synthetase and Mono(ADP-ribosyl)transferasexe2x80x9d, J. of Biol. Chem., 267:1569-75 (1992), identified four compounds that were particularly good inhibitors of PARP, i.e., free of side reactions and applicable to in vivo studies. They were 4-amino-1,8-naphthalimide, 6(5H)- and 2-nitro-6(5H)-phenanthridinones and 1,5-dihydroxyisoquinoline. Comparative studies of the effects of PARP and mono(ADP-ribosyl)transferase from hen heterophils revealed high specificity of most of the potent inhibitors for PARP. Banasik et al., in xe2x80x9cInhibitors and Activators of ADP-ribosylation Reactionsxe2x80x9d, Molec. and Cell. Biochem., 138:185-97 (1994), also described a number of potent Inhibitors of PARP that are specific for PARP as opposed to mono(ADP-ribosyl)transferase.
The method of preventing neural tissue damage resulting from ischemia and reperfusion injury or neurodegenerative diseases in a mammal in accordance with the invention comprises administering to the mammal a therapeutically effective amount of a selective inhibitor of poly(ADP-ribose) polymerase (PARP). In this way, whatever biochemical mechanism or combination of mechanisms that is, in fact, responsible for the excessive PARP activation that often accompanies vascular stroke or other neurodegenerative diseases, the resulting neurotoxicity can be controlled directly and effectively.